Cardiac hypertrophy secondary to chronic pressure or volume overload is manifested by impaired contractile function which in advanced stages of decompensation presents clinically as congestive heart failure (CHF). While tissue environmental factors like decreased coronary vasodilator reserve, altered chamber geometry and interstitial fibrosis have been recognized to contribute to overall myocardial dysfunction, the focus of the current proposal is on abnormalities in excitation-contraction (EC) coupling at the myocyte level in 2 models of CHF: renovascular hypertensive and post-infarct cardiomyopathies. The GOALS are to detect differences in cytosolic free calcium concentration ([Ca]c) dynamics during an EC cycle in control and diseased single myocytes; and to correlate changes in [Ca]c dynamics with alterations in mechanical activity. The OVERALL HYPOTHESIS is that reduced Ca2+ availability during an EC cycle accounts for systolic dysfunction in post-infarct cardiomyopathy but decreased Ca2+ sequestration/extrusion during relaxation accounts for diastolic dysfunction in renovascular hypertensive cardiomyopathy. The SPECIFIC AIMS include: (1) correlate changes in [Ca]c dynamics (measured with fura-2) with alterations in mechanical activity (measured with hybrid digital optical processor) in single myocytes freshly isolated from control and cardiomyopathic hearts; (2) establish sarcomere shortening/lengthening velocity vs log viscosity relationships to define contractile behaviors of myocytes isolated from control and diseased hearts, this will help determine whether systolic or diastolic dysfunction in the working heart persists at the isolated myocyte level; (3) correlate reduction/increase in 3H-PN200-110 binding site density (measured in both cell and isolated sarcolemma) with changes in whole cell Ca2+ current density (measured with patch-clamp); (4) evaluate cellular Ca2+ transporting system defects by measuring kinetics of Na+-Ca2+ exchange, CaATPase activity in SL membrane vesicles and net Ca2+ uptake by sarcoplasmic reticulum (SR) in digitonin- permeabilized myocytes; (5) estimate SR Ca2+ release during an EC cycle by measuring ryanodine-sensitive increases in [Ca]c at 50-100 msec after depolarization; (6) evaluate effects of B-agonists and antagonists on [Ca]c dynamics and contractile activity in control and diseased myocytes; and (7) evaluate effects of various pharmacological agents used to treat CHF on [Ca]c dynamics and contractile activity. Understanding of cellular mechanisms of CHF will allow design of rational therapy.